Cynomolgus prostate specific antigen

ABSTRACT

Isolated polynucleotides encoding Cynomolgus monkey prostate specific antigen and polypeptides obtainable from the polynucleotides and uses are disclosed.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application No.60/575,079, filed 27 May 2004, entitled, “Cynomolgus Prostate SpecificAntigen,” the entire contents of which is incorporated herein byreference.

FIELD OF THE INVENTION

The present invention relates to Cynomolgus monkey prostate specificantigen and its uses.

BACKGROUND OF THE INVENTION

Cancer is a serious disease that afflicts one in four people. In thelast fifty years, there have been significant improvements in the earlydetection of cancer, as well as the development of a number of therapiesto treat cancer. Therapies include surgery to remove primary tumors, andsublethal radiation and chemotherapy to treat disseminated disease.While these treatments have resulted in apparent cures for manypatients, the treatments can be quite debilitating and are still oftenineffective at preventing death from this disease.

Prostate cancer is the second leading cause of cancer-related death inmen. Approximately 180,000 men will be diagnosed with prostate cancereach year and 40,000 succumb to the disease each year. Prostate tumorcells have a low proliferation rate and do not respond to standardchemotherapies, which are most toxic to the most rapidly dividing cellsin the body. Instead, prostate cancer can be treated surgically, withradiation therapy or hormonal therapy. Surgery and radiation therapy canlead to undesirable side effects, such as incontinence and impotence.The disease can often be successfully managed with hormonal therapy,which starves the tumor cells for required growth factors. However,eventually all tumors treated in this way become androgen-independentand there is no effective treatment beyond that point.

Treatment of cancer with active immunotherapy has shown promise in manypreclinical models, and in a few clinical trials as an alternative oradjunct to surgery, radiation or chemotherapy. The goal of activeimmunotherapy is to create a therapeutic immune response againsttumor-specific antigens, which then targets tumor cells for destruction.However, most tumor antigens are self-antigens, to which the patient istolerant. Indeed, central and peripheral tolerance mechanisms areexpected to hamper the generation of effective immunity against tumorsthat express self-antigens.

One way to solve the problem of how to break tolerance against a givenself-antigen is to use a closely related gene or protein from adifferent species as an immunogen. This type of immunization is alsoknown as xenogeneic immunization and its potency lies in either therandom creation of heteroclitic epitopes in the xenogeneic sequenceswith enhanced binding capacity to MHC class I antigens and/or thepresence of strong helper epitopes within the xenogeneic sequence. Forexample, injection of plasmid DNA encoding a xenogeneic differentiationantigen is a powerful means to induce antibody and T-cell responses tootherwise poorly immunogenic self-antigens. This xenogeneic approach hasbeen shown to work for a variety of cancer models using mouse or ratsequences by inducing active immunity to several different types ofgenes, including angiogenesis genes (Liu et al., Blood 102:1815-23,2003), membrane glycoproteins (Wolchok et al., Cancer Immun. 1, 9-18(2001); Sioud and Sorensen D., Eur. J. Immunol. 33, 38-45 (2003)), andintegrins (Lou et al., Immunol. Invest. 31, 51-69 (2002)).

Prostate tumors and some breast malignancies express prostate specificantigen (PSA), also known as kallikrein 3 (KLK3), on their surface. PSAis well known as a serum marker for prostate cancer; increasing serumlevels of PSA typically correlate well with the severity of the disease.It is unclear if PSA has a role in the etiology of prostate cancer;various reports have indicated that PSA could either enhance or inhibittumorigenicity. Several cytotoxic T-lymphocyte (CTL) epitopes for PSAhave been described for the HLA A2 and A3 haplotypes; identification ofthese epitopes support the possibility of generating therapeutic in vivoCTL by vaccination.

In fact, use of DNA encoding human and mouse prostate-specific membraneantigen (PSMA) has been tested in phase I clinical trials in patientswith recurrent prostate cancer. See Wolchok et al., Semin. Oncol. 30,659-66 (2003). These authors have also shown in pre-clinical studiesthat use of xenogeneic DNA (e.g., injection of human PSMA DNA into mice)is an absolute requirement to overcome immunologic tolerance. However, aneed still exists to improve current vaccine strategies by treatinghuman disease with a more closely related PSA antigen to stimulateanti-PSA immunity by xenogeneic immunization.

BRIEF DESCRIPTION OF THE DRAWING

FIG. 1 shows the nucleotide and deduced amino acid sequence offull-length Cynomolgus monkey PSA.

FIG. 2 shows an amino acid sequence comparison of Cynomolgus monkey,Human and Rhesus monkey PSA.

SUMMARY OF THE INVENTION

One aspect of the invention is an isolated polynucleotide comprising apolynucleotide having the sequence shown in SEQ ID NO: 1 or acomplementary sequence, fragment or variant thereof.

Another aspect of the invention is an isolated polynucleotide comprisinga polynucleotide having the sequence shown in SEQ ID NO: 2 or acomplementary sequence, fragment or variant thereof.

Another aspect of the invention is an isolated polynucleotide comprisinga polynucleotide having the sequence shown in SEQ ID NO: 4 or acomplementary sequence, fragment or variant thereof.

Another aspect of the invention is an isolated polynucleotide comprisinga polynucleotide encoding the amino acid sequence shown in SEQ ID NO: 3or a complementary sequence, fragment or variant thereof.

Another aspect of the invention is an isolated polynucleotide comprisinga polynucleotide encoding the amino acid sequence shown in SEQ ID NO: 5or a complementary sequence, fragment or variant thereof.

Another aspect of the invention is an isolated polypeptide comprising apolypeptide having the sequence shown in SEQ ID NO: 3.

Another aspect of the invention is an isolated polypeptide comprising apolypeptide having the sequence shown in SEQ ID NO: 5.

Another aspect of the invention is an isolated full-length humanprostate specific antigen consisting of the amino acid sequence shown inSEQ ID NO: 3.

Another aspect of the invention is an isolated mature human PSAconsisting of the amino acid sequence shown in SEQ ID NO: 5.

DETAILED DESCRIPTION OF THE INVENTION

All publications, including but not limited to patents and patentapplications, cited in this specification are herein incorporated byreference as though fully set forth.

As used herein, the term “DNA vaccines” or “nucleic acid vaccines”denotes compositions useful for the direct in vivo introduction of DNAencoding an antigen into tissues of a subject for expression of theantigen by tissue cells. DNA vaccines are described in, e.g.,International Patent Publications WO 95/20660 and WO 93/19183.

As used herein, the term “nucleic acid adjuvant” means a nucleotidesequence coding for a protein or protein fragment that enhances animmune response to an antigen.

The present invention provides isolated Cynomolgus monkey (Macacafascicularis) PSA polypeptides and polynucleotides. The invention alsoprovides a full-length Cynomolgus monkey PSA having the amino acidsequence set forth in SEQ ID NO: 3 and the polynucleotide encoding itincluding, but not limited to, the polynucleotide having the sequenceset forth in SEQ ID NO: 1 or 2 or their complementary sequences.Full-length Cynomolgus PSA is predicted to have a 24 residue N-terminalleader sequence (SEQ ID NO: 6). The invention also provides a matureCynomolgus monkey PSA lacking the leader sequence and having the aminoacid sequence set forth in SEQ ID NO: 5 and the polynucleotide encodingit including but not limited to, the polynucleotide having the sequenceset forth in SEQ ID NO: 4 or its complementary sequences. The inventionfurther provides for equivalent fragments and variants of Cynomolgusmonkey PSA, as well as encoding or complementary nucleic acids, vectorscomprising a Cynomolgus monkey PSA or fragments or variants, host cellscontaining such vectors and methods of making and methods of use of suchCynomolgus PSA, vectors or host cells.

A “fragment” is a polypeptide having an amino acid sequence that is partof but not all of any amino acid sequence of any polypeptide of theinvention where the fragment contains residues 70 and/or 79 of thefull-length form (equivalent to residues 21 and/or 46 of the matureform). Fragments can include, e.g., truncation polypeptides having aportion of an amino acid sequence as shown in amino acid sequence shownin SEQ ID NO: 3 or 5, or of variants thereof, such as a continuousseries of residues that includes a heterologous amino- and/orcarboxy-terminal amino acid sequence. Degradation forms of thepolypeptides of the invention produced by or in a host cell are alsoincluded. Other exemplary fragments are characterized by structural orfunctional attributes such as fragments that comprise alpha-helix oralpha-helix forming regions, beta-sheet or beta-sheet forming regions,turn or turn-forming regions, coil or coil-forming regions, hydrophilicregions, hydrophobic regions, alpha-amphipathic regions,beta-amphipathic regions, flexible regions, surface-forming regions,substrate binding regions, extracellular regions and high antigenicindex regions.

Specific exemplary fragments include residues 141 to 163 of the matureform that encode peptides that can bind human Class I molecules. SeeCorreale et al., J. Natl. Cancer Inst. 89, 293-300 (1997) and Xue etal., The Prostate 30, 73-78 (1997). Other exemplary fragments includeresidues 16-25, 42-50, 48-56 and 75-83. Further exemplary fragmentsinclude an isolated polypeptide comprising an amino acid sequence havingat least 10, 15, 20, 30, 40, 50 or 100 contiguous amino acids from theamino acid sequence set forth in SEQ ID NO: 3 where the fragmentcontains residues 45 and/or 70 of the full-length form, or an isolatedpolypeptide comprising an amino acid sequence having at least 10, 15,20, 30, 40, 50 or 100 contiguous amino acids from the amino acidsequence set forth in SEQ ID NO: 5 where the fragment contains residues21 and/or 46 of the mature form.

A “variant” polypeptide is a Cynomolgus PSA polypeptide or fragment inwhich amino acid substitutions, insertions, deletions or combinationsthereof have been made. Naturally occurring, modified or atypical aminoacids can be used for substitutions and insertions. A variantpolynucleotide is a polynucleotide encoding variant polypeptides.Variant polypeptides of the invention elicit an immune response to PSAin a host.

The polynucleotides of the invention are useful for preparing acomposition having an isolated polynucleotide encoding an antigenicdeterminant of Cynomolgus monkey PSA and a promoter controllingexpression of the polynucleotide. The composition can further include anisolated polynucleotide encoding a nucleic acid adjuvant such asinterleukin-18 (IL-18) and a promoter polynucleotide controllingexpression of the IL-18. These compositions can be used to elicit animmune response to a cancer-associated tumor protein in a mammal and areuseful as nucleic acid vaccines for the treatment and/or prophylaxis ofcertain cancers or other tumor-related pathologies.

Antigenic determinants useful in the invention are obtained or derivedfrom Cynomolgus monkey PSA. The tumor antigens could also be mutated toenhance their immunogenicity. Examples of how the antigen genes could bemodified to effect a more robust immune response to the antigen proteininclude changes that affect antigen gene expression levels, such asaddition of intron sequences, alteration or removal of signal sequencesrequired for secretion or optimization of codons for improvedtranslation. In addition, the antigen gene could be modified tointroduce changes to the translated product of the gene, such asaddition of ubiquitination signals for degradation, addition ofsubcellular compartment targeting sequences, addition of molecularchaperone sequences, and optimization of CTL epitope sequences. Theantigen genes could be fused together to increase immunogenicity. TheCTL/helper epitopes could be linked together, or inserted as part ofanother molecule, such as an immunoglobulin molecule. Nucleotidesencoding at least one antigenic determinant of the molecules disclosedabove are useful in the invention. Further, sequences complementary toany of the polynucleotides disclosed above are also useful in thecompositions of the invention.

Compositions of the invention including these antigenic determinants areuseful for the treatment of any cancer where PSA is uniquely expressed,over-expressed or associated with the presence of tumors caused by thecancer. These cancers include, but are not limited to, prostate,including hormone-refractory prostate cancer (HRPC), and breast cancer.

The invention also provides a mature polypeptide coding sequence or afragment thereof in reading frame with another coding sequence producedsynthetically or derived from another species, such as a sequenceencoding a leader or secretory sequence, a pre- or pro- orprepro-protein sequence. The polynucleotides of the invention may alsocontain at least one non-coding sequence, such as transcribed but nottranslated sequences, termination signals, ribosome binding sites, mRNAstabilizing sequences, introns and polyadenylation signals. Thepolynucleotide sequences may also contain additional sequences encodingadditional amino acids. These additional polynucleotide sequences may,for example, encode a marker sequence such as a hexa-histidine peptide,as described in Gentz et al., Proc. Natl. Acad. Sci. (USA) 86, 821-824(1989) or the HA peptide tag as described in Wilson et al., Cell 37, 767(1984) which facilitate the purification of fused polypeptides.

The invention also relates to vectors that comprise a polynucleotide orpolynucleotides of the invention, host cells that are geneticallyengineered with vectors of the invention and the production ofpolypeptides of the invention by recombinant techniques. Cell-freetranslation systems can also be employed to produce such proteins usingRNAs derived from the DNA constructs of the invention.

For recombinant production of the polypeptides of the invention, hostcells can be genetically engineered to incorporate expression systems orportions thereof and polynucleotides of the invention. Introduction of apolynucleotide into a host cell can be effected by methods well known tothose skilled in the art from laboratory manuals such as Davis et al.,Basic Methods in Molecular Biology, 2^(nd) ed., Appleton & Lange,Norwalk, Conn. (1994) and Sambrook et al., Molecular Cloning: ALaboratory Manual, 3^(rd) ed., Cold Spring Harbor Laboratory Press, ColdSpring Harbor, N.Y. (2001). These methods include calcium phosphatetransfection, DEAE-Dextran mediated transfection, microinjection,cationic lipid-mediated transfection, electroporation, transduction,scrape loading, ballistic introduction and infection.

Representative examples of hosts include Archaea cells; bacterial cellssuch as streptococci, staphylococci, enterococci, E. coli, streptomyces,cyanobacteria, B. subtilis and S. aureus; fungal cells such asKluveromyces, Saccharomyces, Basidomycete, Candida albicans orAspergillus; insect cells such as Drosophila S2 and Spodoptera Sf9;animal cells such as CHO, COS, HeLa, C127, 3T3, BHK, 293, CV-1, Bowesmelanoma and myeloma; and plant cells, such as gymnosperm or angiospermcells.

A great variety of expression systems can be used to produce thepolypeptides of the invention. Such systems include chromosomal-,episomal- and virus-derived vectors such as vectors derived frombacterial plasmids, bacteriophage, transposons, yeast episomes,insertion elements, yeast chromosomal elements, baculoviruses, papovaviruses such as SV40, vaccinia viruses, adenoviruses, fowl pox viruses,pseudorabies viruses, picronaviruses and retroviruses and vectorsderived from combinations thereof, such as cosmids and phagemids. Theexpression system constructs may contain control regions that regulateor cause expression. Generally, any system or vector suitable tomaintain or propagate polynucleotides and/or to express polypeptides ina host may be used for expression. An appropriate DNA sequence may beinserted into the expression system by any of a variety of techniqueswell known to those skilled in the art, such as, e.g., those set forthin Sambrook et al., supra.

In eukaryotic expression systems, polypeptides of the invention can besecreted into the lumen of the endoplasmic reticulum or extracellularenvironment by inclusion of appropriate secretion signals such as asignal peptide or leader sequence. These signals may be heterologous orendogenous to Cynomolgus PSA such as the signal sequence having theamino acid sequence shown in SEQ ID NO: 6 (predicted).

The polypeptides of the present invention may also be produced bychemical synthesis such as solid phase peptide synthesis on an automatedpeptide synthesizer, using known amino acid sequences or amino acidsequences derived from the DNA sequence of the polynucleotides of theinvention. Such techniques are well known to those skilled in the art.

Polypeptides of the invention can be recovered and purified fromrecombinant cell cultures by well-known methods including ammoniumsulfate or ethanol precipitation, acid extraction, high-performanceliquid chromatography, anion or cation exchange chromatography,phosphocellulose chromatography, hydrophobic interaction chromatography,affinity chromatography, hydroxyapatite chromatography and lectinchromatography. Well-known techniques for refolding protein may beemployed to regenerate an active conformation when the protein isdenatured during isolation and/or purification.

The polynucleotides and polypeptides of the invention comprising atleast one epitope of Cynomolgus PSA can be used to produce polyclonal ormonoclonal antibodies. Techniques for making murine, chimeric, humanizedand fully human monoclonal antibodies using protein or nucleic acidimmunization are known to those skilled in the art.

The polynucleotides and polypeptides of the invention are also usefulfor assaying a medium for the presence of a substance that modulates PSAprotein function by affecting the binding of a Cynomolgus PSA protein toserum proteins. Examples of modulators include polypeptides or smallorganic molecules.

For use as DNA vaccines, the polypeptides of the invention could becontained within one or more cellular delivery vectors such as plasmids,mammalian viruses, bacteria or mammalian cells having appropriateregulatory and control elements as are well known to those skilled inthe art. For example, expression of the tumor antigen and nucleic acidadjuvant polynucleotide sequences could be under the control of asuitable promoter such as the human cytomegalovirus immediate early(HCMV IE) promoter or dihydrofolate reductase promoter, and apolyadenylation (polyA) signal such as the SV40 late, SV40 early polyAsignal or a synthetic polyA sequence. An intron may be included forenhanced expression, such as the HCMV IE intron A or natural intronsfrom the antigen or adjuvant genes.

An exemplary plasmid useful with the polypeptides of the inventioncontains an E. coli origin of replication, an aph(3′)-1a kanamycinresistance gene, HCMV immediate early promoter with intron A, asynthetic polyA sequence and a bovine growth hormone terminator. Anotherexemplary plasmid contains an E. coli origin of replication, anant(4′)-1a kanamycin resistance gene, Rous sarcoma virus long terminalrepeat sequences, HCMV immediate early promoter and an SV40 late polyAsequence.

Examples of suitable viruses that can act as recombinant viral hosts forthe polypeptides of the invention include vaccinia, canarypox, andadenovirus, as are known in the art. Various genetically engineeredvirus hosts (“recombinant viruses”) can also be used. Viral cellulardelivery vectors containing the compositions of the invention canpromote a suitable immune response that targets activation of Blymphocytes, helper T lymphocytes, and cytotoxic T lymphocytes.

A preferred recombinant virus for use with the compositions of theinvention is vaccinia virus (International Patent Publication WO87/06262; Cooney et al., Proc. Natl. Acad. Sci. USA 90, 1882-1886(1993); Graham et al., J. Infect. Dis. 166, 244-252 (1992); McElrath etal., J. Infect. Dis. 169, 41-47 (1994)). In another embodiment,recombinant canarypox can be used (Pialoux et al., AIDS Res. Hum.Retroviruses 11, 373-381 (1995), erratum in AIDS Res. Hum. Retroviruses11, 875 (1995); Andersson et al., J. Infect. Dis. 174, 977-985 (1996);Fries et al., Vaccine 14, 428-434 (1996); Gonczol et al., Vaccine 13,1080-1085 (1995)). Another alternative is defective adenovirus oradeno-associated viruses or retroviruses (Gilardi-Hebenstreit et al., J.Gen. Virol. 71, 2425-2431 (1990); Prevec et al., J. Infect. Dis. 161,27-30 (1990); Lubeck et al., Proc. Natl. Acad. Sci. USA 86, 6763-6767(1989); Xiang et al., Virology 219, 220-227 (1996)). Other suitableviral vectors include attenuated or defective DNA virus, such as but notlimited to herpes simplex virus (HSV) (see, e.g., Kaplitt et al., Molec.Cell. Neurosci. 2, 320-330 (1991)), papillomavirus, Epstein Barr virus(EBV), see, e.g., U.S. Pat. Nos. 5,990,091; 5,766,599; 5,756,103;6,086,890; 6,274,147; 5,585,254; 6,140,114; 5,616,326; 6,099,847;6,221,136; 6,086,891; 5,958,425; 5,744,143; 5,558,860; 5,266,489;5,858,368; 5,795,872; 5,693,530; 6,020,172.

The polypeptides of the invention can be formulated in apharmaceutically acceptable carrier or diluent. A variety of aqueouscarriers may be employed, e.g., 0.4% saline, 0.3% glycine and the like.These solutions are sterile and generally free of particulate matter.These solutions may be sterilized by conventional, well-knownsterilization techniques (e.g., filtration). The compositions maycontain pharmaceutically acceptable auxiliary substances as required toapproximate physiological conditions, such as pH adjusting and bufferingagents. The concentration of the polypeptides of the invention in suchpharmaceutical formulation can vary widely, i.e., from less than about0.5%, usually at or at least about 1% to as much as 15 or 20% by weightand will be selected primarily based on fluid volumes, viscosities andother factors, according to the particular mode of administrationselected. Further, plasmids containing the polypeptides of the inventioncould also be formulated in microparticles or with lipid, buffer orother excipients.

The polypeptides of the invention can also be formulated with adjuvantsthat could aid delivery of DNA, maintain its integrity in vivo orenhance the immunogenicity of the vaccine. Chemical adjuvants caninclude compounds or mixtures that enhances the immune response to anantigen. A chemical adjuvant can serve as a tissue depot that slowlyreleases the antigen and also as a lymphoid system activator thatnon-specifically enhances the immune response (Hood et al., Immunology,2nd ed., (1984), Benjamin/Cummings, Menlo Park, Calif., p. 384).Adjuvants include, but are not limited to, complete Freund's adjuvant,incomplete Freund's adjuvant, saponin, mineral gels such as aluminumhydroxide, surface active substances such as lysolecithin, pluronicpolyols, polyanions, peptides, oil or hydrocarbon emulsions, keyholelimpet hemocyanins, dinitrophenol, and useful human adjuvants such asBCG (Bacillus Calmette-Guerin) and Corynebacterium parvum. Selection ofan adjuvant depends on the subject to be vaccinated. Preferably, apharmaceutically acceptable adjuvant is used. For example, a vaccine fora human should avoid oil or hydrocarbon emulsion adjuvants, includingcomplete and incomplete Freund's adjuvant. One example of an adjuvantsuitable for use with humans is alum (alumina gel). In a specificembodiment, compositions of the invention are administeredintramuscularly in alum. Alternatively, the compositions of theinvention can be administered subcutaneously, intradermally,intraperitoneally, intramuscularly or via other acceptable vaccineadministration routes.

The polypeptides of the invention can be temporally administered indifferent orders or administered in different places in the body at thesame time. The polypeptides of the invention could also be delivered bydirect injection into muscle, skin, lymph node, or by application tomucosal surfaces. In a specific embodiment, polypeptides of theinvention are provided intramuscularly. Other potential modes ofdelivery would include injection of DNA, followed by electroporation toenhance cellular uptake and expression of DNA or administration by agene gun or a similar device. For screening anti-tumor activity of seraor cells from an individual immunized with a polypeptide of theinvention, any suitable screening assay can be used, as is known in theart.

The polypeptides of the invention, when in a pharmaceutical preparation,can be present in unit dose forms. The appropriate therapeuticallyeffective dose can be determined readily by those of skill in the art. Adetermined dose may, if necessary, be repeated at appropriate timeintervals selected as appropriate by a physician during the treatmentperiod.

The polypeptides of the invention can be lyophilized for storage andreconstituted in a suitable carrier prior to use. This technique hasbeen shown to be effective with conventional protein preparations andart-known lyophilization and reconstitution techniques can be employed.

The present invention will now be described with reference to thefollowing specific, non-limiting examples.

EXAMPLE 1 Isolation, Cloning and Sequencing of Cynamolgus Monkey PSAGene

RNA was purified from Cynomolgus prostate tissue (Cambrex Bio ScienceWalkersville Inc, Walkersville, Md.) using Trizol reagent (Invitrogen,Carlsbad, Calif.) according to the manufacturer's instructions. Reversetranscription reactions were carried out using Superscript II ReverseTranscriptase kit and were primed using an oligo dT primer. Forpolymerase chain reaction (PCR), a gene-specific forward primer thatannealed to the translation initiation site was paired with a reverseprimer that annealed to conserved regions within the 3′ untranslatedregions of the Human and Rhesus PSA mRNAs (SEQ ID NOs: 11 and 12, Table1, 5′ PSA and 3′ PSA). The first ATG is located at residues 19-21 of the5′ PSA primer shown in Table 1. The 5′ PSA primer anneals to thetranslation initiation site with a Bam HI site (underlined in Table 1)engineered on the 5′ end and the 3′ PSA primer anneals to a conservedregion within the 3′ untranslated region with a Bam HI site engineeredon the 3′ end. PCR was performed using 100 ng of each primer with 100 ngcDNA template under the following conditions: 30 cycles of 94° C. 30seconds, 60° C. 1 minute, 68° C. 1 minute, followed by one cycle of 68°C. for 5 minutes. Ten percent of the PCR reactions were analyzed onagarose gels, and revealed a band of approximately 1000 bp (data notshown). PCR products were purified using Qiaquick PCR purification(Qiagen, Valencia, Calif.). Approximately 60 ng of each product wasdirectly sequenced using the Big Dye Terminator Cycle Sequencing Kit (PEApplied Biosystems, Foster City, Calif.), followed by analysis on aPrism ABI377 automated DNA sequencing apparatus. PCR fragments weresequenced using 20 ng of 5′ PSA seq and 3′ PSA seq primers correspondingto nucleotides 383-406 and 653-676, respectively (SEQ ID NOs: 13 and 14,Table 1). In addition, specific PCR products were also subcloned andsequenced. All PSA gene results were confirmed from prostate tissue fromat least two Cynomolgus monkeys.

The sequences of the fragments were compared using Vector NTi software(Invitrogen, Frederick, Md.) to the Genbank database and aligned withHuman and Rhesus PSA sequences (Genbank accession #M21896 and #X73560,respectively). The cynomolgus PSA sequence was also BLAST searchedagainst available embl and other sequence databases. BLASTP, TBLASTN andBLASTN searches were performed and no identical records were found.

To obtain the complete sequence of the 5′ and 3′ untranslated regions ofthe cynomolgus PSA gene, rapid amplification of cDNA ends (RACE) wasemployed. Total RNA was extracted from the prostates of at least twoadult male Cynomolgus monkeys and the 5′ and 3′ untranslated regions ofthe mRNA transcript was amplified by ligase-mediated RACE reactionsusing the 5′ and 3′ RACE primers shown in Table 1 corresponding tonucleotides 49-72 and 782-805, respectively (SEQ ID NOs: 15 and 16).

TABLE 1 Cynomolgus PSA gene cloning and sequencing oligonucleotides.Name Sequence 5′ PSA 5′-CTGGATCCCTGTGCCACCATGTGG-3′ (SEQ ID NO: 11)3′ PSA 5′-CCGGATCCTGCTGATTTCTTTTCC-3′ (SEQ ID NO: 12) 5′ PSA seq5′-CCAGCCACGACCTCATGCTGCTCC-3′ (SEQ ID NO: 13) 3′ PSA seq5′-CCCCAGAATCACCCGACAGGTGC-3′ (SEQ ID NO: 14) 5′ RACE5′-CGTCACGGACAGGGTGAGGAAGAC-3′ (SEQ ID NO: 15) 3′ RACE5′-GGAAGTGGAGCCAGGACACCATCA-3′ (SEQ ID NO: 16)

Four clones from three different monkeys were sequenced and aligned. Thenucleotide and deduced amino acid sequence of full-length Cynomolgusmonkey PSA cDNA is shown in FIG. 1 and SEQ ID NOs: 1 and 2. In FIG. 1,the translated amino acid sequences of the coding region are numberedand shown above the nucleotide sequence. The nucleotide residues arenumbered on the left. The putative polyadenylation signal is in boldtype.

Sequencing analysis of the cDNA fragments showed that the Cynomolgus PSAmRNA consists of a short 5′ noncoding region of about 33 bases (residues1 to 33 of SEQ ID NO: 1), an open reading frame of 783 bases (residues34 to 816 of SEQ ID NO: 1), corresponding to 261 amino acids (SEQ ID NO:3) and a 3′ untranslated region of 660 bases (residues 817 to 1476 ofSEQ ID NO: 1). The first 24 amino acids of Cynomolgus PSA are expectedto include the signal sequence (SEQ ID NO: 6). The amino terminus ofmature PSA is predicted to be the isoleucine shown as residue 1 inFIG. 1. The polynucleotide sequence and amino acid sequence of themature 237 amino acid form of Cynomolgus PSA is shown in SEQ ID NOs: 4and 5, respectively.

EXAMPLE 2 Sequence Comparisons of Primate PSA Genes

Protein sequence comparisons of the Cynomolgus PSA sequence to theGenbank database of Human and Rhesus PSA cDNAs are shown in FIG. 2.Residues that differ between the species are shown in bold andhighlighted. Each species encodes a mature protein predicted to be 237amino acids in length. The comparisons revealed that the Cynomolgus PSAamino acid sequence was 89.7% identical to Human PSA (27/261 residuedifferences) (FIG. 2). The Rhesus amino acid sequence was 90% identicalto the human gene (26/261 residue differences). The Cynomolgus andRhesus amino acid sequences differed at only two amino acids, namelyresidues 21 and 46 of the mature form, for 99.2% identity.

The nucleotide sequences of the three genes were also compared for thecoding region. There is approximately 93.5% identity between Human andthe Cynomolgus and Rhesus sequences (data not shown). Only threenucleotide residues differ in this region between the two primate cDNAs,giving 99.6% identity. Like the Human and Rhesus proteins, CynomolgusPSA also has a serine at position 183, which has been shown to beresponsible for the cleavage specificity of the serine proteases athydrophobic amino acids (Lundwall, A. and Lilja, H. FEBS Letters 214,317-322 (1987)).

The 5′ untranslated regions (UTR) were identical between Rhesus andCynomolgus monkeys. The sequence of the Cynomolgus 5′ UTR was eightbases longer than the human sequence (data not shown).

There were several differences in the 3′ UTRs between the species.First, the Cynomolgus 3′ UTR is 75 bases shorter than the rhesus 3′UTRwith a 72 base pair deletion located at bases 405/406 from the TGA stopcodon of the Cynomolgus sequence. Besides these deletions, there areonly three nucleotide differences in the 3′UTR between the species. Asexpected, there were more differences between the Human and Cynomolgus3′UTR. First, there is a 54 base pair deletion in the human sequencecorresponding to bp 34-87 of the Cynomolgus sequence. In addition, thereare 4 base pair additions in the Cynomolgus sequence and one base pairdeletion in addition to the 72 base pair deletion compared to the humansequence. There are also 31 base pair differences between the species(data not shown).

EXAMPLE 3 Analysis of PSA Expression in Cynomolgus Tissues

To investigate the RNA expression pattern of Cynomolgus PSA inCynomolgus tissues, RT-PCR analysis was performed using RNA purifiedfrom different tissues. Total RNA from the kidney, lung, testis, thymusand prostate was isolated and cDNA was synthesized as described inExample 1. No DNA template was used as a negative control fornon-specific amplification. Isolated total RNA was used as a templatefor first strand cDNA synthesis using Superscript II reversetranscriptase and an oligo dT primer. 100 ng of cDNA was subjected toPCR amplification as described above. The PSA gene was amplified usingthe 5′ PSA (SEQ ID NO: 11) and 3′ PSA (SEQ ID NO: 12) primers to yield a1000 bp PSA-specific product. β-actin was amplified as a loading controlusing Cynomolgus-specific forward (SEQ ID NO: 17) and reverse (SEQ IDNO: 18) primers; a 216 bp product was amplified. The results indicatedthat PSA mRNA was only detected in the prostate tissue and not in thekidney, lung, testis or thymus (data not shown).

The present invention now being fully described, it will be apparent toone of ordinary skill in the art that many changes and modifications canbe made thereto without departing from the spirit or scope of theappended claims.

1. An isolated polynucleotide comprising a polynucleotide having thesequence shown in SEQ ID NO:1 or the full complementary sequencethereof.
 2. An isolated polynucleotide comprising a polynucleotidehaving the sequence shown in SEQ ID NO:2 or the full complementarysequence thereof.
 3. An isolated polynucleotide comprising apolynucleotide having the sequence shown in SEQ ID NO:4 or the fullcomplementary sequence thereof.
 4. An isolated polynucleotide comprisinga polynucleotide encoding the amino acid sequence shown in SEQ ID NO:3or the full complementary sequence thereof.
 5. An isolatedpolynucleotide comprising a polynucleotide encoding the amino acidsequence shown in SEQ ID NO:5 or the full complementary sequencethereof.
 6. A vector comprising the isolated polynucleotide of claim 1,2, 3, 4 or
 5. 7. An isolated host cell comprising the vector of claim 6.8. A process for producing a polypeptide comprising culturing the hostcell of claim 7 under conditions sufficient for production of thepolypeptide.